The family of Ras signal transduction proteins are proto-oncogenes that are frequently mutated in human cancers. They are encoded by three ubiquitously expressed genes. These proteins are GTPases which function as molecular switches regulating pathways involved in cell proliferation, differentiation and survival.
It seems that all mammalian cells express these closely related Ras proteins. Under normal circumstances, Ras proteins are regulated by guanine nucleotide exchange factors (GEF) that promote GDP dissociation and GTP binding, and GTPase-activating proteins (GAP) that stimulate the intrinsic GTPase activity of Ras to switch off signaling.
Aberrant Ras function is associated with hyperproliferative development disorders and cancers and in tumors it is associated with a single mutation typically at codon 12, 13 or 61(1). Mutations at these conserved sites favors GTP binding and produces constitutive activation of Ras. All Ras isoforms share sequence identity in all of the regions that are responsible for GDP/GTP binding, GTPase activity, and effector interactions, suggesting a functional redundancy. Nevertheless, it is becoming increasingly apparent that Ras proteins have isoform-specific functions. These functional differences are most likely associated with the unique C-terminal hypervariable region (HVR) in each isoform, which is thought to modulate the Ras membrane interaction to specify distinctive localizations in organelles and signaling nanoclusters.
Early analysis of Ras isoform mutational status in cancer revealed varying incidences of Ras mutations in different tumor types, and specific associations of individual Ras isoforms with particular cancers. Despite the relatively small sample sizes used in those analyses, investigators were able to identify strong trends. For example, K-Ras was shown to be the most frequently mutated isoform in most cancers, with 90% of pancreatic tumors harboring K-Ras mutations. In contrast, N-Ras mutations were more strongly associated with hematopoietic tumors and with thyroid cancer. With the advent of large-scale tumor profiling and data sequencing databases, researchers can now perform deeper analyses of Ras mutational spectra. The Catalog of Somatic Mutations in Cancer (COSMIC) is the most comprehensive database in human tumor mutations currently available.
With respect to NRAS, almost 60% of NRAS-positive tumors harbor mutations at codon 61, compared with 35% at codon 12. In contrast, 80% of K-Ras mutations occur at codon 12, whereas very few mutations are observed at codon 61. Ras codons 12, 13 and 61 can each be converted to six other amino acids via single-base substitutions. However, more than 60% of the total mutations for each isoform are accounted for by only three of the 18 potential mutations across the codons. Since, for example, DNA sequences of wild-type, Q61K, Q61L and Q61R differ from each other only by a single nucleotide, it is still very difficult to detect these mutations with high specificity and sensitivity.
As the type of mutation in NRAS may be important for the type of treatment chosen by the physician after a clinical diagnosis of cancer, it is important to provide a simple and reliable test for NRAS that can be performed in a clinical laboratory.
Such a test is provided in the present application. The present invention is based on a unique primer design that allows identification of NRAS mutations in DNA extracted from biological samples. The set of oligonucleotides according to the present invention allows detection of each specific NRAS polynucleotide isoforms, i.e. also of mutant isoforms carrying SNP's that are relevant in cancer. The test of the present invention will be helpful in diagnostic tests and allows with high accuracy to determine which mutations are present in nucleic acid isolated from a sample of an affected individual.